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Detecting Individual Extracellular Vesicles Using a Multicolor in Situ www.nature.com/scientificreports OPEN Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow Received: 28 April 2016 Accepted: 09 September 2016 cytometric readout Published: 29 September 2016 Liza Löf 1, Tonge Ebai1, Louise Dubois2, Lotta Wik1, K. Göran Ronquist2, Olivia Nolander1, Emma Lundin1, Ola Söderberg1, Ulf Landegren1 & Masood Kamali-Moghaddam1 Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells. However, flow cytometric analysis of extracellular vesicles (EVs), and the proteins present on their surfaces has been hampered by the small size of the EVs – in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present, and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer. By targeting sets of proteins on the surface that are specific for distinct classes of EVs, the method allows for selective recognition of populations of EVs in samples containing more than one type of EVs. The method presented herein opens up for analyses of EVs using flow cytometry for their characterization and quantification. Cells have the capacity to release different kinds of vesicles into the extracellular space, collectively named extra- cellular vesicles (EVs). EVs can be grossly subdivided into three subclasses based on their biogenesis and in order of increasing sizes; exosomes, microvesicles, and apoptotic bodies. Exosomes (including prostasomes derived from epithelial cells of the prostate gland1) are the smallest subclass, typically ranging in size between 40–100 nm in diameter, although larger sizes have been reported1–3. They arise through two invagination events; a first invagination of the plasma membrane produces “early endosomes” that mature into “late endosomes.” The latter undergo multiple invaginations, creating a multivesicular body whose membrane fuses with the plasma membrane from the inside, releasing its exosomal content into the extracellular space through exocytosis2,3. Microvesicles have sizes of 200–1,000 nm, and are the result of shedding from the plasma membrane – hence they are also denoted “shedding vesicles”4. Apoptotic bodies are the largest extracellular vesicles released during apop- tosis and with diameters from one to a few μ m2,5. EVs may exert pleiotropic biological functions by trafficking between cells. They can influence the microenvironment by transporting bioactive molecules, including proteins, lipids and RNA6–8. EVs have been implicated in physiological and pathological processes such as inflammation, immune disorders, and cancer9,10. Prostasomes have been found to be elevated in blood plasma from prostate cancer patients, where the levels of prostasomes in blood plasma may reflect the aggressiveness of the disease11. Among the proteins most frequently identified in EVs are the tetraspanin family members CD9, CD63, and CD81, the small actin-binding protein cofilin-1, the heat shock proteins Hsp70, Hsp90, and enzymes involved in energy metabolism such as enolase, aldolase A, phosphoglycerate kinase 1 and glyceraldehyde 3-phosphate dehydrogenase12. A number of methods are currently available to detect and measure intact EVs. Nanoparticle tracking analysis (NTA) measures EV concentration and size based on scattered light or fluorescence (Malvern Instruments Ltd. United Kingdom). Resistive pulse sensing (RPS) determines the absolute sizes of the EVs in suspension using a specialized instrument, qNano, (Izon Science Ltd. New Zealand). A common limitation here is clogging of the system13. Enzyme-linked immunosorbent assays (ELISA) are used to analyze the proteins in a sample and are conventionally used to analyze EVs in clinical settings. This method, however, requires large 1Department of Immunology, Genetics & Pathology, Science for Life Laboratory, Uppsala University, SE-751 08 Uppsala, Sweden. 2Department of Medical Sciences, Clinical Chemistry, Uppsala University, SE-751 85 Uppsala, Sweden. Correspondence and requests for materials should be addressed to M.K.-M. (email: [email protected]) SCIENTIFIC REPORTS | 6:34358 | DOI: 10.1038/srep34358 1 www.nature.com/scientificreports/ amounts of EVs and is hampered by the time-consuming isolation process13,14. 4PLA developed in our lab is another sensitive method for detection of intact EVs. 4PLA requires binding by a total of five different antibodies in order to produce detectable signals, ensuring high specificity11. The PLA technology has been used to sensi- tively detect complex targets in body fluids, also in the presence of high concentrations of proteins in monomeric forms. EVs (prostasomes) were detected in the presence of abundant free protein molecules in plasma11 and amyloid beta protofibrils could be sensitively measured in cerebrospinal fluid despite a large molar excess of free amyloid beta monomers15. In 2014 Im et al. described the nano plasmonic exosome (nPLEX) assay, a label-free quantitative assay for analysis of exosomes. The assay is based on nanohole arrays, which can be utilized to profile the proteins on the surface of exosomes or in exosome lysates16. EVs are also investigated for their contents, and many assays exist that are devoted to study e.g. mRNAs and miRNA transported in the vesicles, but these methods are beyond the scope of the present investigation. Despite the plethora of techniques to find and characterize EVs in biofluids the need remains for improved methods. Flow cytometry-based methods are attractive for detecting EVs, both for qualitative and quantitative characterization. However, the small size of the EVs represents a considerable challenge, making individual EVs difficult or impossible to distinguish from background by conventional flow cytometry where the lower size detection threshold is around 500 nm17. Another problem is the paucity of antigen molecules present on the surface of the EVs due to their limited size, rendering detection with fluorophore-coupled antibodies very challenging. These difficulties are often resolved by using bead-based assays, or by lipid-membrane staining and bulk measurements18. In bead-based assays, EVs are captured on beads, and stained for surface markers using fluorophore-coupled antibodies. This is a satisfactory alternative for detecting the presence of EVs in a sample, since each bead can bind several EVs and stain for surface antigens, thus providing sufficiently strong signals to be detected in the flow cytometer. However, the current bead-based assays are not suitable for identification of individual EVs since the beads may carry a large number of EV particles sharing one antigen recognized by the capturing antibody. To address limitations of flow cytometric detection of EVs, we applied the in situ proximity ligation assay (in situ PLA)19,20 in a method we refer to as ExoPLA. By using multicolor in situ PLA21 we label the EVs in three colors, in assays that depend on binding of individual EVs by a total of five different antibodies, providing out- standing assay specificity. Utilizing the signal amplification feature of in situ PLA, the signal from individual EVs is sufficiently strong to be detected well above the background. In this method EVs are captured on beads, fol- lowed by specific detection, washes, and then release from the beads before signal amplification via rolling circle amplification (RCA)19. The release of the EVs from the beads prior to signal amplification and detection serves to permit analysis of individual EVs in a flow cytometer, and to avoid the co-localization of signals generated from different EV on same bead particle. In situ PLA utilizes PLA probes; antibodies coupled to DNA oligonucleotides. When two PLA probes are brought in proximity, a DNA circle can be obtained through two ligation reactions, templated by the antibody-bound oligonucleotides. This DNA circle then serves as a template for RCA. Detection oligonucleotides coupled to fluorophores are hybridized to the growing RCA product. Each detected protein gives rise to an RCA product with several hundred fluorophores, ensuring strong detection signals for individual EVs. Since EVs from different cell sources may reflect the identity of the originating cell22, a method to distinguish different populations of EVs using conventional flow cytometry may permit enhanced resolution through anal- ysis of individual EVs. As previously shown, prostasomes represent promising biomarker candidates in prostate cancer11. To test if ExoPLA is sensitive and specific enough to find prostasomes in a complex matrix, we spiked prostasomes isolated from seminal fluid in female blood plasma, demonstrating the ability of ExoPLA to accu- rately detect prostasomes against a background of all resident proteins and EVs in female plasma. Results Overview of ExoPLA. The ExoPLA technique uses capturing beads, allowing immobilized EVs to be stained through multiplex in situ PLA, for subsequent release before amplification and analysis of individual EVs by flow cytometry. Streptavidin-modified magnetic beads bind biotinylated oligonucleotides, which then immobilize oligonucleotide-conjugated capturing antibodies via hybridization (Fig. 1). The hybridization oligonucleotides contain uracil residues
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